BioMicroCenter:Illumina Sequencing

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ILLUMINA MASSIVELY PARALLEL SEQUENCING

The MIT BioMicro Center has one high-throughput Solexa Genome Analyzers 2.0 (Illumina), which is currently being used for a variety of applications, including ChIP-Seq, miRNA sequencing and expression sequencing. The next-generation sequencer can process up to 16 samples per week, with a data yield of approximately 10 million reads per sample. Read lengths can be any length upto 36 bases. Longer runs (upto 72nt) should be available in early 2009.

Illumina sequencing works by binding randomly fragmented DNA to an optical flowcell . Templates are sequenced by incorporating fluorescently labeled nucleotides in a “Sequencing-By-Synthesis” reaction. A detailed description of this process can be found here: http://illumina.com/pages.ilmn?ID=203

The system consists of a cluster generation station, a Paired-End module, and a Genome Analyzer, all of which work in concert to generate and analyze flowcells. An overview of the Illumina Genome Analyzer system can be found at the Illumina website: http://illumina.com/pages.ilmn?ID=204

Applications

Illumina currently provides reagents and support for a five major sequencing applications:

• ChIP Seq
• RNA Seq
• Small RNA Sequencing
• Genome sequencing and resequencing
• Paired End Sequencing
• Multiplexed Sequencing

Other DNA Sequencing applications: The following applications have been published but do not yet have kits from Illumina.

• Genotyping: Protocols are being developed for detection of SNPs, chromosomal rearrangements and other genotyping applications.
  
• Genome Wide Nuclear Run-on Analysis (GRO-seq): This method allows for the detection of nascent transcripts directly associated with the genome. GRO-seq was developed by John Lis at Cornell University. Reference: http://www.sciencemag.org/cgi/content/abstract/1162228

Data Analysis

Each lane of the flow cell should produce sequence from between 2 and 5 million DNA fragments. Understanding this data often requires a significant investment in informatics. This is complicated by the fact that many applications require entirely different interpretations of the data. As part of our sequencing service we provide many of the early steps of bioinformatics for different applications. Further data processing can be arranged on a collaborative basis as resources are available. For more information, check out the links below:

• Illumina pipeline - How it works! -- under construction
• Illumina pipeline output formats
• Bioinformatic Consulting

Pricing

Illumina sequencing is currently available only for labs associated with the BioMicro Center Core departments. Full pricing information is available at our price list.

ILLUMINA SEQUENCING	CORE LAB/MIT	Non-MIT	unit	Notes
Single End (36nt)	$860	$1,800	per lane	Includes quality control (RT-PCR and BioAnalyzer), sequencing, genome alignment and data storage of Firecrest files for 2 yrs.
Paired End (36+36nt)	$1,450	$3,000	per lane
Add'l 36nt	$300	$555	per lane
Sample Multiplexing	$50	$100	per 10 samples	Combined over the whole order. Only applies to samples combined by BCM technicians.
Add'l Repriming	$300	$600	per flow cell	Uses 6nt. Cost is divided by number of lanes requiring repriming. Repriming is required for standard Illumina multiplexing.

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Protocols

Protocols for all of the supported technologies can be found by visiting the Protocols page

QC

Quality control is very important to help optimize the number of reads and quality of data produced. For information on QC methods and protocols please visit the Sequencing Quality Control page

MIT Core Collaboration

All samples run on the Illumina sequencer are run in batches of 7 or 8 samples. In order to optimize our throughput, we are trying out a novel strategy that allows us to move partial flowcells between the various centers at MIT. For users with less then 4 samples, their samples may be moved between the BioMicro Center, the Whitehead Institute Center for Genome Technologies and the Koch Institute Biopolymer Center. Samples will be moved only to fill out runs and to expedite processing. The Centers are committed to working together to maintain consistent quality between the different cores so you should see no difference wether your samples are run in BioMicro or at one of our sister centers. A list of samples in line for transfer can be found Here. Transfers are only available for members of the MIT community.

All questions about Illumina Sequencing can be directed to Kevin Thai at kthai@mit.edu.

Initial page written by Summeet Gupta at the WI-CGT